Generation Of Neurospheres Research Articles

Generation and Differentiation of Primary Neurospheres from Zebrafish Neural Stem Cells

Generation and Differentiation of Primary Neurospheres from Zebrafish Neural Stem Cells

Generation and Differentiation of Primary Neurospheres from Zebrafish Neural Stem Cells

Generation and Differentiation of Primary Neurospheres from Zebrafish Neural Stem Cells

P02.09.A THE PLASTICITY AND SURVIVAL OF GLIOBLASTOMA STEM CELLS DEPEND ON GD3, WHOSE INHIBITION IS SYNERGISTIC WITH RADIO-CHEMOTHERAPY

Abstract BACKGROUND Glioblastoma is the most frequent and aggressive primary brain tumor in adults. No curative treatment is available, and the identification of relevant therapeutic targets is an unmet medical need. Our team previously demonstrated that A2B5 positive cells have a stemness behavior, with self-renewal, multipotency ability, and tumor initiation ability in vivo. A2B5 is recognizing different gangliosides, including GT3 and its precursor GD3. However, A2B5 is an IgM, that cannot be used directly as a therapeutic strategy.Then, our aim was to demonstrate that GD3 was a relevant and actionable target for glioblastoma stem cells (GSC). MATERIAL AND METHODS We used the GCS lines we already derived from fresh patient samples. Then, we sorted GD3 positive cells from patient samples to derive new GCS lines and explore GD3 properties. We downregulated the GD3 synthase (ST8Sia1) by using Sh. The tumor cells were orthotopically engrafted in nude mice. RESULTS First, we showed that GD3 was expressed by 15 to 20% of cells from patient tumor bulks but not by normal brain. Then, we showed that 60% to 90% of GSC expressed GD3. Importantly, this fraction dropped between 20 to 40% after cell differentiation. We sorted GD3 positive cells from patient samples and showed their ability to exhibit stemness properties including the generation of neurospheres (expressing stem cell markers like SOX2 and nestin), clonogenicity, and tumorigenicity after orthotopic engraftment. We also demonstrated that GD3+ cells had plasticity properties with differentiation and dedifferentiation abilities depending on their culture medium. Then, by using shST8SIA1 in GSC lines, we showed that the reduction of ST8Sia1/GD3 was associated with a decreasing size of spheres, and reduced clonogenicity and migratory capacities. Moreover, the reduction of ST8Sia1/GD3 expression increased the temozolomide and radiotherapy sensitivity in vitro with a synergistic effect. Finally, the reduction of ST8Sia1/GD3 expression in U87 cell line was associated with an increasing mouse survival after orthotopic engraftment in nude mice. CONCLUSION Taken together, our results strongly suggest that GD3 and ST8SIA1 are essential for GSC properties. GD3 appears to be an interesting and actionable stem cell marker, opening promising therapeutic development for GBM patients.

Neural Tissue Engineering with Rat Adipose-Derived Mesenchymal Stem Cells: The Role of an Injectable, Resorbable Hydrogel Scaffold Derived from Oxidized Alginate and Gelatin.

The central nervous system has limited regeneration potential. The multipotency of adipose-derived mesenchymal stem cells (ADMSC) makes them an ideal autologous cell source for the regeneration of neural tissues. However, the likelihood of their differentiation into unwanted cell lineages when transplanted into a hostile injury environment is a serious disadvantage. Transplanting predifferentiated cells via an injectable carrier may aid in site-specific delivery for better survival of cells. Here, we focus on identifying an appropriate injectable hydrogel system that favors stem/progenitor cell attachment and differentiation for neural tissue engineering. An injectable composition of the hydrogel, derived from alginate dialdehyde (ADA) and gelatin, was formulated for this purpose. This hydrogel promoted proliferation/differentiation of ADMSCs to neural progenitors, visualized from the generation of prominent neurospheres and stage-specific expression of a neural progenitor marker (nestin, day 4), an intermittent neuronal marker (β-III tub, day 5), and a mature neuronal marker (MAP-2, day 8) with neural branching and networking (>85%). The differentiated cells also expressed the functional marker synaptophysin. There was no negative impact on stem/progenitor cell survival (>95%) or differentiation (∼90%) as compared to two-dimensional (2D) culture. Addition of appropriate quantities of asiatic acid specific for neural niche supported cell growth and differentiation without affecting cell survival (>90%) and improved neural branching and elongation. Optimized interconnected porous hydrogel niche exhibited rapid gelation (3 min) and self-healing properties mimicking native neural tissue. Both ADA-gelatin hydrogel by itself and that incorporated with asiatic acid were found to support stem/neural progenitor cell growth and differentiation and have potential applications as antioxidants and growth promoters upon release at the cell transplantation site. In short, the matrix itself or incorporated with phytomoieties could serve as a potential minimally invasive injectable cell delivery vehicle for cell-based therapies of neural diseases.

A Robust Pipeline for the Multi-Stage Accelerated Differentiation of Functional 3D Cortical Organoids from Human Pluripotent Stem Cells.

Disordered cellular development, abnormal neuroanatomical formations, and dysfunction of neuronal circuitry are among the pathological manifestations of cortical regions in the brain that are often implicated in complex neurodevelopmental disorders. With the advancement of stem cell methodologies such as cerebral organoid generation, it is possible to study these processes in vitro using 3D cellular platforms that mirror key developmental stages occurring throughout embryonic neurogenesis. Patterning-based stem cell models of directed neuronal development offer one approach to accomplish this, but these protocols often require protracted periods of cell culture to generate diverse cell types and current methods are plagued by a lack of specificity, reproducibility, and temporal control over cell derivation. Although ectopic expression of transcription factors offers another avenue to rapidly generate neurons, this process of direct lineage conversion bypasses critical junctures of neurodevelopment during which disease-relevant manifestations may occur. Here, we present a directed differentiation approach for generating human pluripotent stem cell (hPSC)-derived cortical organoids with accelerated lineage specification to generate functionally mature cortical neurons in a shorter timeline than previously established protocols. This novel protocol provides precise guidance for the specification of neuronal cell type identity as well as temporal control over the pace at which cortical lineage trajectories are established. Furthermore, we present assays that can be used as tools to interrogate stage-specific developmental signaling mechanisms. By recapitulating major components of embryonic neurogenesis, this protocol allows for improved in vitro modeling of cortical development while providing a platform that can be utilized to uncover disease-specific mechanisms of disordered development at various stages across the differentiation timeline. © 2023 Wiley Periodicals LLC. Basic Protocol 1: 3D hPSC neural induction Support Protocol 1: Neural rosette formation assay Support Protocol 2: Neurosphere generation Support Protocol 3: Enzymatic dissociation, NSC expansion, and cryopreservation Basic Protocol 2: 3D neural progenitor expansion Basic Protocol 3: 3D accelerated cortical lineage patterning and terminal differentiation.

Ubiquitin-Specific Protease 22 Promotes Neural Stem Cells Stemness Maintenance and Adult Hippocampal Neurogenesis, Contributing to Cognitive Recovery Following Traumatic Brain Injury

Ubiquitin-specific protease 22 (USP22), a potential marker of cancer stem cells, significantly influences stem cell fate choices. However, its functions in neural stem cells (NSCs) and adult neurogenesis, especially following traumatic brain injury (TBI), remain only partially understood. Here, we found that aberrant USP22 expression could affect NSC proliferation and stemness maintenance, as assessed by the generation of neurospheres, cell counting kit-8 (CCK-8) and immunofluorescence staining in vitro. Moreover, USP22 depletion promotes the differentiation of NSCs, both in vitro and in vivo. In contrast, USP22 overexpression inhibits NSC differentiation into neurons. Interestingly, our data showed that USP22 promotes the proliferation but inhibits the differentiation of NSCs in the dentate gyrus (DG) of the hippocampus soon after TBI. The Morris water maze (MWM) test was adopted to evaluate neurological function, which confirmed that USP22 could improve the learning and memory capacity that was already compromised following TBI. Overall, this study uncovers a potentially novel regulatory role of USP22 in the proliferation and differentiation ability of NSCs, contributing to the hippocampus-dependent cognitive function of TBI mice and may be a novel target for future therapeutic approaches.

Explant culture and multilineage differentiation of amniotic membrane derived stem cells

Amniotic membrane derived stem cells (AMSCs) are reported to have a comparatively higher potency than multipotent stem cells. These cells are shown to have low immunogenicity and no teratogenicity. Among various conventional methods of isolation using enzymes, explant culture method is believed to be an easy and cost-effective way to harvest stem cells. The purpose of this study was to isolate AMSCs from amniotic membrane of rats and to characterize them for multilineage differentiation, including generation of neurospheres to use them later in in-vivo experiments. Amniotic membranes were collected from Wistar rats on 17th day of pregnancy. After processing of the tissues, AMSCs were isolated by the explant culture method and continued to grow until 10th passage. The doubling time was estimated and the cells were analyzed for growth curve parameters at passages 5 and 9. The osteogenic and adipogenic differentiation studies were carried out from the same cells after 3rd passage. Neurospheres generation from AMSCs was performed using neurogenic induction media. The cells were further assessed for their mesenchymal, haemopoietic, and neurogenic marker expressions by immunofluorescence staining and PCR analysis The study suggests that AMSCs isolated through explant culture are reliable stem cells which could generate neurospheres under proper induction conditions and could be a potential candidate to be used on in-vivo neural degeneration models.

In vitro investigation of the differentiation of enteric neural crest-derived cells following transplantation of aganglionic gut in a mouse model.

Cell therapy is a promising approach to treat enteric neuropathies such as Hirschsprung disease (HD). Recent studies have reported that enteric neurons derived from stem cells (ENCCs) can be grafted into the HD colon. Thus, we investigated the migration and generation of enteric neurospheres from SOX10-VENUS+ mice after transplantation into control or Ednrb KO mice, which are a model of HD. Single-cell suspensions were isolated from the fetal guts of SOX10-VENUS+ mice E13.5 and dissociated. These cells were cultured for 7days under non-adherent conditions to generate neurospheres, which were co-cultured with dissociated control or SOX10-VENUS- Ednrb KO mouse gut on E13.5. 4days later, these cells were fixed and the expression of the neuronal marker, Tuj1, was evaluated. Transplanted neurospheres had undergone abundant neuronal migration and differentiation of ENCCs in the control gut compared with the HD gut. The average length and intersections were significantly decreased in HD colon compared with controls (p < 0.05), and a similar pattern was observed in the HD small intestine (p < 0.05). We demonstrated that transplanted ENCCs did not differentiate properly in HD gut. These results highlight the importance of the neuronal environment in the recipient gut for enteric nervous system development.

Retroviral infection of human neurospheres and use of stem Cell EVs to repair cellular damage

HIV-1 remains an incurable infection that is associated with substantial economic and epidemiologic impacts. HIV-associated neurocognitive disorders (HAND) are commonly linked with HIV-1 infection; despite the development of combination antiretroviral therapy (cART), HAND is still reported to affect at least 50% of HIV-1 infected individuals. It is believed that the over-amplification of inflammatory pathways, along with release of toxic viral proteins from infected cells, are primarily responsible for the neurological damage that is observed in HAND; however, the underlying mechanisms are not well-defined. Therefore, there is an unmet need to develop more physiologically relevant and reliable platforms for studying these pathologies. In recent years, neurospheres derived from induced pluripotent stem cells (iPSCs) have been utilized to model the effects of different neurotropic viruses. Here, we report the generation of neurospheres from iPSC-derived neural progenitor cells (NPCs) and we show that these cultures are permissive to retroviral (e.g. HIV-1, HTLV-1) replication. In addition, we also examine the potential effects of stem cell derived extracellular vesicles (EVs) on HIV-1 damaged cells as there is abundant literature supporting the reparative and regenerative properties of stem cell EVs in the context of various CNS pathologies. Consistent with the literature, our data suggests that stem cell EVs may modulate neuroprotective and anti-inflammatory properties in damaged cells. Collectively, this study demonstrates the feasibility of NPC-derived neurospheres for modeling HIV-1 infection and, subsequently, highlights the potential of stem cell EVs for rescuing cellular damage induced by HIV-1 infection.

The RNA helicases DDX5 and DDX17 facilitate neural differentiation of human pluripotent stem cells NTERA2

AimsUnderstanding human neurogenesis is critical toward regenerative medicine for neurodegeneration. However, little is known how neural differentiation is regulated by DEAD box-containing RNA helicases, which comprise a diverse class of RNA remodeling enzymes. Materials and methodsChIP-seq was utilized to identify binding sites of DDX5 and DDX17 in both human pluripotent stem cell (hPSC) line NTERA2 and their retinoic acid-induced neural derivatives. RNA-seq was used to elucidate genes differentially expressed upon depletion of DDX5 and DDX17. Neurosphere assay, flow cytometry, and immunofluorescence staining were performed to test the effect of depletion of the two RNA helicases in neural differentiation. Key findingsWe show here that expression of DDX5 and DDX17 is abundant throughout neural differentiation of NTERA2, and is mostly localized within the nucleus. The two RNA helicases occupy chromatin genome-wide at regions associated with neurogenesis-related genes in both hPSCs and their neural derivatives. Further, both DDX5 and DDX17 are mutually required for controlling transcriptional expression of these genes, but are not important for maintenance of stem cell state of hPSCs. In contrast, they facilitate early neural differentiation of hPSCs, generation of neurospheres from the stem cells, and transcriptional expression of key neurogenic transcription factors such as SOX1 and PAX6 during neural differentiation. Importantly, DDX5 and DDX17 are critical for differentiation of hPSCs toward NESTIN- and TUBB3-positive cells, which represent neural progenitors and mature neurons, respectively. SignificanceCollectively, our findings suggest the role of DDX5 and DDX17 in transcriptional regulation of genes involved in neurogenesis, and hence in neural differentiation of hPSCs.

Generation of Rat Neural Stem Cells to Produce Different Astrocyte Phenotypes.

Striatum-derived neural stem cells have been used to generate a variety of neural cell populations. They are composed of free-floating clusters of clonal neural stem cells, termed neurospheres, and can be expanded under growth factor stimulation in vitro. The multipotent nature of neurospheres means that under certain growth conditions they can differentiate into neuronal and glial progenitors of the central nervous system (CNS).Here, we describe a method for creating a population of astrocytes derived from rat striatum neurospheres, which in turn can be used to generate astrocytes with different reactivity phenotypes. Several methods and techniques are already available for the generation of neurospheres, but the method detailed herein provides an accessible, reproducible protocol for large numbers of astrocyte cultures, that can then be manipulated in an experimental format for further investigation.

Isolation and Culture of Adult Hippocampal Precursor Cells as Free-Floating Neurospheres.

The neurosphere assay is the most widely used in vitro tool to determine the proliferative and differentiation potential of adult neural precursor cells in rodents. Although originally developed for, and predominantly applied to, the growth of embryonic and adult subventricular zone-derived stem cells, hippocampal neurospheres are now routinely cultured by many laboratories. As hippocampal neurospheres are fewer in number, on average smaller in size, and more slowly growing than their ventricular counterparts, the methodology traditionally used to isolate and culture neurospheres from the subventricular zone is not optimal for hippocampal neurosphere growth. Here, we provide a detailed description of an optimized protocol for the microdissection, dissociation, and neurosphere generation from adult hippocampal dentate gyrus tissue. We also outline the protocols required to perform downstream passaging, differentiation, and immunohistological determination of the multipotentiality of hippocampal neurospheres.

Dopamine regulates adult neurogenesis in the ventricular-subventricular zone via dopamine D3 angiotensin type 2 receptor interactions.

Adult neurogenesis is a dynamic and highly regulated process, and different studies suggest that dopamine modulates ventricular-subventricular zone (V-SVZ) neurogenesis. However, the specific role of dopamine and the mechanisms/factors underlying its effects on physiological and pathological conditions such as Parkinson's disease (PD) are not fully understood. Recent studies have described counter-regulatory interactions between renin-angiotensin system (RAS) and dopamine in peripheral tissues and in the nigrostriatal system. We have previously demonstrated that angiotensin receptors regulate proliferation and generation of neuroblasts in the rodent V-SVZ. However, possible interactions between dopamine receptors and RAS in the V-SVZ and their role in alterations of neurogenesis in animal models of PD have not been investigated. In V-SVZ cultures, activation of dopamine receptors induced changes in the expression of angiotensin receptors. Moreover, dopamine, via D2-like receptors and particularly D3 receptors, increased generation of neurospheres derived from the V-SVZ and this effect was mediated by angiotensin type-2 (AT2) receptors. In rats, we observed a marked reduction in proliferation and generation of neuroblasts in the V-SVZ of dopamine-depleted animals, and inhibition of AT1 receptors or activation of AT2 receptors restored proliferation and generation of neuroblasts to control levels. Moreover, intrastriatal mesencephalic grafts partially restored proliferation and generation of neuroblasts observed in the V-SVZ of dopamine-depleted rats. Our data revealed that dopamine and angiotensin receptor interactions play a major role in the regulation of V-SVZ and suggest potential beneficial effects of RAS modulators on the regulation of adult V-SVZ neurogenesis.

Generation of UCiPSC-derived neurospheres for cell therapy and its application

BackgroundNeural stem cell (NSC) therapy remains one of the most potential approaches for the treatment of neurological disorders. The discovery of human induced pluripotent stem cells (hiPSCs) and the establishment of hiPSC-derived human neural stem cells (hiNSCs) have revolutionized the technique of cell therapy. Meanwhile, it is often required that NSCs are stored and transported to a long distance for research or treatment purposes. Although high survival rates could be maintained, conventional methods for cell transportation (dry ice or liquid nitrogen) are inconvenient and expensive. Therefore, the establishment of a safe, affordable, and low-cost strategy to store and transport easily accessible hiPSCs and hiNSCs, with characteristics that match fetal hNSCs, is incredibly urgent.MethodsWe reprogrammed human urinary cells to iPSCs using a non-integrating, virus-free technique and differentiated the iPSCs toward iNSCs/neurospheres and neurons, under Good Manufacturing Practice (GMP)-compatible conditions. The pluripotency of iPSCs and iNSCs was characterized by a series of classical methods (surface markers, karyotype analysis, and in vitro as well as in vivo differentiation capabilities, etc.).ResultsHere, our results showed that we successfully generated hiNSCs/neurospheres from more available, non-invasive, and more acceptable urinary cells by a virus-free technique. Next, we demonstrated that the iNSCs differentiated into mature cerebral cortical neurons and neural networks. Interestingly, hiNSCs survived longer as neurospheres at ambient temperature (AT) than those cultured in a monolayer. Within 7 days approximately, the neural viability remained at > 80%, while hiNSCs cultured in a monolayer died almost immediately. Neurospheres exposed to AT that were placed under standard culture conditions (37 °C, 5% CO2) recovered their typical morphology, and retained their proliferation and differentiation abilities.ConclusionsIn this study, we provided a simple method for the storage of NSCs as neurospheres at AT as an alternative method to more costly and inconvenient traditional methods of cryopreservation. This will enable hiNSCs to be transported over long distances at AT and facilitate the therapeutic application of NSCs as neurospheres without any further treatment.

Quantitative approach to numbers and sizes: Generation of primary neurospheres from the dorsal lateral ganglionic eminence of late embryonic mice.

Background: The neurosphere assay is a powerful in vitro tool to investigate neural stem cells in the dorsal lateral ventricle (dLGE). In the dLGE, metrics of sizes and numbers of neurospheres generated using this assay has not been completely characterized. The objective of this protocol is to provide a stepwise method from a single isolation that predicts the average number of neurospheres generated and to estimate an approximation of its sizes after several days in vitro. The advantage of this protocol is that no expensive and specialized equipment is needed for tissue isolation. Estimates about the numbers and sizes of neurospheres will provide investigators with quantitative data to advise on how much starting dLGE tissue is required to generate the appropriate number of spheres for the implementation of downstream applications, including immunocytochemistry, self-renewal and differentiation assays. Methods: Our method is based on a simple dissection technique, where tissue surrounding the dorsal lateral ventricle from a single mouse embryo is trimmed away to enrich for neural stem cell and progenitor populations. Following this dissection, tissue is mechanically dissociated by trituration. Cells are then cultured in media containing epidermal growth factor and other supplements to generate healthy primary neurospheres. Results: Using this approach, we found reproducible number of primary neurospheres after 7 days in vitro (DIV). Furthermore, we observed that this method yields an average range of neurospheres sizes greater than 50 μm, but less than 100 μm after 7 DIV. Lastly, using an anti-GFAP antibody, we show that these neurospheres can be stained, confirming their use in future immunocytochemistry studies. Conclusions: Future use of this protocol provides metrics on the generation of primary neurospheres that will be useful for further advances in the area of stem cell biology.

Identification of neurospheres generated from human dental pulp stem cells in xeno-/serum-free conditions

IntroductionCell-based therapies require an emerging alternative treatment using easily harvested cell sources. Neural stem cells derived from various tissues, including brain, bone marrow, skin and retina can give rise to both neurons and glial cells. Recently, human dental pulp stem cells (DPSCs) and stem cells from human exfoliated deciduous teeth (SHED) were demonstrated to have mesenchymal stem cell-like abilities such as self-renewal and multi-lineage differentiation, including neuron and glial cells. Moreover, DPSCs and SHED show a higher proliferation rate and a higher number of population doublings compared with adult bone marrow stromal stem cells. Therefore, DPSCs are a useful source that can be applied in cell replacement therapy for various neurological disorders. Generally, the conventional culture methods for DPSCs have used serum, therefore the undefined components in culture medium may complicate investigations of the molecular mechanisms that control the self-renewal and differentiation of DPSCs. However, neural stem cells proliferate to form ‘neurospheres’ in suspension in vitro in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). No study to date has obtained neurospheres from DPSCs in serum-free conditions in primary culture. Thus, the aim of this study was to establish a method for the proliferation and neural differentiation of DPSCs in xeno- and serum-free conditions in primary culture.MethodsDPSCs were obtained from the dental pulp of wisdom teeth from healthy individuals (18–41 years old) and cultured in conventional medium containing 15% fetal bovine serum and xeno-/serum-free medium. We evaluated the proliferation of DPSCs, neurosphere generation, and neural differentiation under xeno-/serum-free conditions by flow cytometry, immunohistochemistry, and real-time polymerase chain reaction.ResultsIn proliferation medium without xeno/serum, DPSCs can proliferate and generate neurospheres, however, the neurospheres had limited self-renewal ability. Under differentiation conditions, class III β-tubulin (TUBB3) and microtubule-associated protein (MAP2) were more significantly expressed in neurospheres derived from DPSCs in xeno-/serum-free culture conditions than in DPSCs in conventional culture conditions.ConclusionsOur result demonstrated that neurosphere generation from DPSCs in xeno-/serum-free culture may be an accessible source for clinical cell replacement therapies for neuronal degenerative diseases.

Neonatal hypoxia-ischemia in rat elicits a region-specific neurotrophic response in SVZ microglia

BackgroundRecent findings describe microglia as modulators of neurogenesis in the subventricular zone (SVZ). SVZ microglia in the adult rat are thought to adopt a neurotrophic phenotype after ischemic stroke. Early postnatal microglia are endogenously activated and may therefore exhibit an increased sensitivity to neonatal hypoxia-ischemia (HI). The goal of this study was to investigate the impact of cortico-striatal HI on the microglial phenotype, function, and gene expression in the early postnatal SVZ.MethodsPostnatal day (P)7 rats underwent sham or right-hemispheric HI surgery. Microglia in the SVZ, the uninjured cortex, and corpus callosum were immunohistochemically analyzed at P10, P20, and P40. The transcriptome of microdissected SVZ and cortical microglia was analyzed at P10 and P20, and the effect of P10 SVZ microglia on neurosphere generation in vitro was studied.ResultsThe microglial response to HI was region-specific. In the SVZ, a microglial accumulation, prolonged activation and phagocytosis was noted that was not observed in the cortex and corpus callosum. The transcriptome of SVZ microglia and cortical microglia were distinct, and after HI, SVZ microglia concurrently upregulated pro- and anti-inflammatory as well as neurotrophic genes. In vitro, microglia isolated from the SVZ supported neurosphere generation in a concentration-dependent manner.ConclusionsMicroglia are an inherent cellular component of the early postnatal SVZ and undergo developmental changes that are affected on many aspects by neonatal HI injury. Our results demonstrate that early postnatal SVZ microglia are sensitive to HI injury and display a long-lasting region-specific response including neurotrophic features.

Abstract A058: Dielectric characterization of glioblastoma cancer stem cells

Abstract Glioblastoma (GBM) is the most aggressive type of primary brain tumor, characterized by the intrinsic resistance to chemotherapy due to the presence of a highly aggressive Cancer Stem Cell (CSC) sub-population. The coexistence in the same mass of bulk and CSCs stuck at different stages of differentiation and genetic backgrounds prevents counteracting all cells with single treatments. Through the application of a Dielectrophoretic force (DEP) we provide a successful CSC isolation from a mixed cell population, exploiting their physical properties. Using FACS, the expression of specific stem cell and differentiation markers (CD133, Nestin, Sox1, Sox2, GFAP, Doublecortin (DCX) and Ki67) were analyzed in 17 primary cell lines of GBM. CD133 expression was also exploited as a reliable marker for the identification of the CSC subpopulation and thus used for sorting CD133+ and CD133- GBM primary cells by flow cytometry techniques. CD133+ and CD133- cells were characterized for their ability to self-renew and form neurospheres. In all experiments, CD133+ cells have shown a significant increased efficiency of neurosphere generation that was accompanied by a higher self-renewal potential as measured by the limiting dilution assay (LDA) in vitro. The characterization and isolation of patient-derived GBM cells by FACS allowed constituting a cell library with three distinct cell subpopulation types: CD133+/CD133-/Total population, that have been characterized for their specific Electromagnetic (EM) signature. Determination of EM signature was performed using a sensor that applies electromagnetic fields in the MHz regime to selectively electromanipulate cells with dielectrophoresis (DEP) and allows the monitoring of the induced cell motion generated by the field. By scanning the frequency range of interest, the cell DEP signature and the cross over frequency (frequency for which the DEP force becomes null) were identified. We found that CD133+ and CD133- cell display a significantly different crossover frequency. In particular, CD133+ cells are characterized by a lower cross over frequency than both CD133- and total population. This data suggest a non obvious correlation between crossover frequency and CSCs phenotype which would be potentially exploited to set up a label free system able to efficiently identify and physically separate subpopulation of cells enriched for CSC characteristics. Citation Format: Roberta Bortolozzi, Elena Mariotto, Elena Rampazzo, Elena Porcù, Sofiane Saada, Thomas Provent, Fabrice Lalloué, Arnaud Pothier, Giampietro Viola, Luca Persano. Dielectric characterization of glioblastoma cancer stem cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A058. doi:10.1158/1535-7163.TARG-19-A058

Quantitative approach to numbers and sizes: Generation of primary neurospheres from the dorsal lateral ganglionic eminence of late embryonic mice

Background: The neurosphere assay is a powerful tool to study neural stem cell biology. The objective of this protocol is to create a simple and rapid approach to generate neurospheres from the dorsal lateral ganglionic eminence of late embryonic (day 17) mice. This method predicts the average number of neurospheres and provides an approximation of its expected size after 7 days in vitro. Characterization of numbers and sizes will provide investigators with quantitative data to advise on the implementation of downstream applications, including immnocytochemistry, self-renewal and differentiation assays. Methods: Our method is based on a simple dissection technique, where tissue surrounding the dorsal lateral ventricle from a single mouse embryo is trimmed away to enrich for neural stem cell and progenitor populations. Following this dissection, tissue is mechanically dissociated by trituration. Cells are then cultured in media containing epidermal growth factor and other supplements to generate healthy primary neurospheres. Results: Using this approach, we found reproducible number of primary neurospheres after 7 days in vitro. Furthermore, we found this method yields different sizes of neurospheres. Lastly, using an anti-GFAP antibody, we confirm that these neurospheres can be used for immunocytochemistry studies. Conclusions: Future use of this protocol provides metrics on the generation of neurospheres that will be useful for further advances in the area of stem cell biology.

Generation of Neurospheres from Mixed Primary Hippocampal and Cortical Neurons Isolated from E14-E16 Sprague Dawley Rat Embryo.

Primary neuron culture is an essential technique in the field of neuroscience. To gain deeper mechanistic insights into the brain, it is essential to have a robust in vitro model that can be exploited for various neurobiology studies. Though primary neuron cultures (i.e., long-term hippocampal cultures) have provided scientists with models, it does not yet represent the complexity of brain network completely. In the wake of these limitations, a new model has emerged using neurospheres, which bears a closer resemblance to the brain tissue. The present protocol describes the plating of high and low densities of mixed cortical and hippocampal neurons isolated from the embryo of embryonic day 14-16 Sprague Dawley rats. This allows for the generation of neurospheres and long-term primary neuron culture as two independent platforms to conduct further studies. This process is extremely simple and cost-effective, as it minimizes several steps and reagents previously deemed essential for neuron culture. This is a robust protocol with minimal requirements that can be performed with achievable results and further used for a diversity of studies related to neuroscience.